ICES mar. Sei. Symp., 199: 459-467. 1995

Genetic differentiation in Berryteuthis magister from the North Pacific

O. N. Katugin

Katugin, O. N. 1995. Genetic differentiation in Berryteuthis magister from the North Pacific. - ICES mar. Sei. Symp., 199: 459-467.

Berryteuthis magister is a widespread quasibenthic commercial squid from the North Pacific. Intraspecific genetic differentiation was determined by allozyme electrophore­ tic analysis. Eighteen sample lots (2100 individuals) from geographically separated North Pacific regions were subjected to allozyme electrophoretic analysis using a total of 14 enzymes and unidentified ganglion protein spectra with polymorphic zones. Four loci with variant allele frequencies greater than 0.05 were found to be useful for population studies. No significant violations of the Hardy-Weinberg equilibrium were found at any loci in the samples. There was no evidence of genetic differences between sexes. Analysis of genetic differentiation using Wright’s F-statistics, cluster analysis of genetic distances, and contingency chi-square analysis suggested that there are popu­ lation differences between squids from the three major geographical localities: the Sea of Japan, the Kurile-Komandor region, and the Gulf of Alaska. Genetic divergence between squid from the Kurile-Komandor part of the species range probably reflects subpopulation differentiation of local stocks from successive generations.

O. N. Katugin: Pacific Research Institute o f Fisheries and Oceanography (TINRO), Vladivostok, 690 600, Russia [tel: (+7) 4232 25 7790, fax: (+7) 4232 25 7783],

been investigated electrophoretically. This family is con­ Introduction sidered to be the most abundant group of cephalopods in During the last two decades biochemical genetic tech­ the subarctic waters of the Pacific Ocean where it pre­ niques based on electrophoretic separation of multiple sumably originated and diverged (Nesis, 1973). It com­ protein forms, especially enzymes, have uncovered a prises about 17-19 species in three genera (Nesis, 1982): great source of “cryptic” (non-morphological) variation Gonatus, Gonatopsis, and Berryteuthis. that exists in natural populations of a variety of taxa. Berryteuthis magister has a wide distribution range; Most polymorphisms at individual enzyme loci have adult squid dwell on the continental slope and in the appeared to be interpretable from a genetic standpoint near-shelf waters of the Sea of Japan, along the Kurile because of co-dominant inheritance of most of the allelic Chain, in the Sea of Okhotsk, across the Bering Sea, and isoenzymes (allozymes). Frequencies of electrophoreti- through the Gulf of Alaska down to California. The cally detectable allelic variants have been widely used in species is quasibenthic, and passes through three stages, animal population structure analysis, particularly of or ecological phases, in its orthogenesis: (1) planktonic invertebrates. Though many molluscan species have hatchlings and young juveniles in the epipelagic layers, been investigated by this approach, few studies have (2) micronectonic juveniles (descending phase), and (3) involved cephalopods. Most of the cephalopod studies nectobenthic adults (Kubodera, 1982). The species has a concerned myopsid squids of the family Loliginidae lifespan of about one or two years (Naito et al., 1977', (Ally and Keck, 1978; Christofferson et a i, 1978; Nat- Yuuki and Kitazawa, 1986; Okutani, 1988; Nesis, 1989). sukari etal., 1986; Augustyn and Grant, 1988; Carvalho The spawning period extends over nearly two months or andLoney, 1989; Garthwaite etal., 1989; Brierley etal., more, followed by the death of the spent squid. Biologi­ 1993b; Yeatman and Benzie, 1993), and oegopsid squids cal and fisheries data suggest that intraspecific variability of the family Ommastrephidae (Murata et al., 1985; exists in B. magister. Discrete groups with uncertain Smith et al., 1987; Carvalho et al., 1992; Brierley et al., populational status have been revealed by the analysis of dorsal mantle lengths, body weights, and stages of 1993a; Kim, 1993). Oegopsid squids of the family Gonatidae have not maturity (Fedorets, in press). Geographically isolated 460 O. N. Katugin ICES mar sd Symp 1W (ms)

Table 1. Location, collection dates, sample sizes, and state of maturity for Berryteuthis magister.

Location Coordinates Date N M Sea of Japan 1 Kitajamato Bank 39°50'N 133°44'E Oct., 1987 200 AI Kurile Islands 2 Southern Kuriles 44°30'N 147°50'E Feb., 1990 96 AI 3 Central Kuriles 47°04'N 152°20'E Sep., 1989 100 AI 4 Central Kuriles 47°02'N 152°14'E Feb., 1990 223 AR 5 Central Kuriles 47°01'N 152°16'E May, 1990 93 AR 6 Central Kuriles 47°02'N 152°37'E May, 1991 214 AR 7 Northern Kuriles 49°48’N 156°28'E Aug., 1988 99 AR 8 Northern Kuriles 49°43'N 155°20'E Aug., 1989 104 AR 9 Northern Kuriles 49°17'N 155°39'E Feb., 1990 100 AR 10 Northern Kuriles 49°28'N 155°36'E Sep., 1990 88 AI 11 Northern Kuriles 49°41'N 156°22'E Nov., 1991 63 AI Western Bering Sea 12 Komandor Islands 55°27'N 165°10'E Jul., 1988 107 AR 13 Komandor Islands 54°23'N 167°70'E Apr., 1989 100 AM 14 Komandor Islands 54°20'N 167°06'E May, 1989 100 AM 15 Komandor Islands 54°20'N 168°17'E Jan., 1990 94 AM 16 Komandor Islands 53°23'N 165°17'E May, 1990 95 AM 17 Navarin Bay 61°40'N 176°30'E Sep., 1990 160 AM Gulf of Alaska 18 Gulf of Alaska 57°23'N 155°15'W Mar., 1988 46 AI. AR N no. of specimens in a sample; M - maturity (AI — immature adults; AR = ripening adults; AM = mature adults).

groups within the species are expected for the following reasons: (1) oceanographic conditions differ consider­ ably across the range; (2) certain geographic barriers, such as islands and shallow straits, occur across the range • 1 7 of the squid; (3) spawning grounds are presumably re­ 6 0 " stricted to some deepwater regions, though they have 18 not been determined precisely due to the difficulties of 50" trawling; (4) early life stages concentrate in several dis­ crete North Pacific areas, namely, in the southeastern 40" part of the Okhotsk Sea, in the central southern part of the Bering Sea, in the Gulf of Alaska (Kubodera and Jefferts, 1984), and in the Sea of Japan (Yuuki and Kitazawa, 1986). Since the 1970s the gonate squid B. magister has been the object of a commercial fishery. The annual catch by Figure 1. Locations from which samples of Berryteuthis magis-magis­ ter were taken. Numbers correspond to those in Table 1. the Russian fishing fleet in the waters near the Kurile Islands is about 70000 t (Fedorets, pers. comm.) and has been expanding. In order to establish reasonable com­ mercial management for this squid, it is necessary to Materials and methods determine the number of self-sustaining intraspecific Eighteen sample lots of B. magister were taken from groups, or breeding populations, that make up the scientific research catches during 1987-1990 by bottom species. For this purpose, selected enzymes from mantle trawling (Table 1, Fig. 1). A total of about 2100 squid tissue extracts, and general protein spectra from gang­ were frozen at — 20°C for electrophoretic investigation. lion tissue extracts were studied by electrophoresis in Individual mantle and ganglion tissue samples were starch and polyacrylamide gels. The revealed polymor­ prepared for analysis by standard techniques: about phic genetic loci were used to elucidate intraspecific 1 cm3 of tissue from every animal was homogenized with genetic differentiation in B. magister with regard to its an equal amount of distilled water and centrifuged at population structure. 8000g for 30 min at 0°C. Vertical electrophoresis in 5% ic e s mar. Sd. Symp., 199(1995) Genetic differentiation in Berryteuthis magister from the North Pacific 461 polyacrylamide gels in a continuous TRIS-EDTA Na2- contribution of samples, groups of samples, and individ­ borate (pH 8.4) buffer system (Peacock et al., 1965), and ual loci to the total level of genetic differentiation. horizontal electrophoresis in 14% starch gels in a con­ tinuous TRIS-maleate (pH 7.4) buffer system (Shaw and Prasad, 1970) were used to separate water-soluble pro­ Results teins from individual supernatants. Ganglion general protein spectra were revealed on polyacrylamide gels Eleven enzymes were stained and gave good interpre­ with Coomassie BB (G-250) in 12.5% TCA (Katugin, table electrophoretic banding patterns: glycerophos­ 1991). Enzymatic activity in mantle tissue extracts was phate dehydrogenase (locus Gpd), lactate dehydrogen­ revealed by histochemical staining protocols (Shaw and ase (two loci: Ldh-1, and Ldh-2), NAD-dependent Prasad, 1970; Harris and Hopkinson, 1976). malate dehydrogenase (locus M dh), NADP-dependent Alleles were designated according to the electrophor­ malate dehydrogenase (locus Me), isocitric dehydrogen­ etic mobilities of their products, relative to those of the ase (locus Idh), 6-phosphogluconate dehydrogenase most common allele (which was designated 100). Loci (locus Pgd), phosphoglucose isomerase (locus Pgi), for a particular enzyme were numbered in order of phosphoglucomutase (two loci: Pgm-1, and Pgm-2), decreasing electrophoretic mobility towards the anode, superoxide dismutase (locus Sod), esterase (two loci: and the slowest locus was assigned number 1. A locus Est-1, and Est-2), and acid phosphatase (locus Acp). was considered polymorphic if the frequency of the Fourteen presumptive structural loci were coding for common allele did not exceed 0.95 in at least one sample these enzymes. Electrophoretic variation was observed of squid. at the loci Gpd, Sod, and Est-2, but was not taken into Genotypic frequencies for each highly polymorphic account due to the accepted 95% criterion of polymor­ locus were examined for agreement with Hardy-Wein- phism. Three enzyme loci were sufficiently polymorphic berg expectations using a goodness-of-fit chi-square test. for population genetic analysis: Pgd, Pgm-1, and Mdh. We used the following equation to compare frequencies Four alleles were observed for the 6-phosphogluco- of common alleles between males and females in the nate dehydrogenase (Pgd). Five out of 10 theoretically largest sample of animals (Urbach, 1964): possible phenotypes were detected on polyacrylamide gels. Allozymes encoded by the Pgd had one-banded homozygous and three-banded heterozygous pheno­ t = (Pi) - (p2)/V l/n i + l/n2, types. Phenotypic distributions at the Pgd did not devi­ ate significantly from the Hardy-Weinberg expectations in any of the samples. where t is the coefficient showing significance of differ­ Five alleles were observed at the slow migrating zone ences, (p,) and (p2) are arcsine transformed allelic fre­ of phosphoglucomutase activity (Pgm-1). Eight out of quencies for a particular locus in different sexes, n] and 15 expected phenotypes were detected on starch gel. n2 are doubled number of males and females respect­ Proteins encoded by the Pgm-1 presented one-banded ively. phenotypes in homozygotes and two-banded pheno­ To measure electrophoretically detectable genetic dif­ types in heterozygotes indicating monomeric structure ferentiation between samples we used two coefficients: of the enzyme. No apparent violations of the Hardy- N ei’s (1972) genetic distance Z?N, and Rogers’ (1972) Weinberg proportions were detected for the Pgm-1 in genetic distance 0 R. The first statistic shows the number the samples studied. of codon differences acquired between the two samples We observed one anodal, highly variable zone of (populations) since their divergence from the common malate dehydrogenase activity, corresponding to one ancestor. The second one shows the differences between polymorphic locus M dh with four co-dominant alleles. the two samples (populations) in space, but is not pro­ Eight out of 10 possible phenotypes were found. Homo­ portional to the number of gene substitutions (Nei, zygotes were one-banded, and heterozygotes had three- 1987). Both statistics were calculated using allele fre­ banded phenotypes. Phenotypic distributions at the quencies of only four polymorphic loci. We chose an Mdh were consistent with Hardy-Weinberg expec­ UPGM A (unweighted pair-group method with arith­ tations in all samples, except sample 17 from Navarin metic mean) distance matrix method for constructing Bay (x2 = 10.82; d.f. = 3; p < 0.05). Significant value is phenograms (Sokal and Michener, 1958). Heterogen­ due to deficiency of homozygotes. eity of allelic frequencies among sample collections was Nearly 20 discrete bands were resolved on the general examined by the chi-square statistic (Workman and Nis- protein spectra for the ganglion tissue. Four zones of the wander, 1970). Wright’s fixation indices or F-statistics spectra were variable between individuals, but only one (Wright, 1965, 1978; Nei, 1977) were used to evaluate zone presented a genetically interpretable picture of the magnitude of intersample divergence, and to assess variation. This zone of anodal slowly migrating unidenti- 462 O. N. Katugin ICES mar sd Symp m (m5)

Alleles

Collection no. Pgd(100) Pgm-1 (100) M dh(U5) Mdh(lOO) Ugp(lOO) 1 0.980 0.797 0.136 0.460 0.864 2 0.942 0.848 0.078 0.593 0.814 3 0.920 0.850 0.055 0.565 0.760 4 0.933 0.875 0.117 0.547 0.793 5 0.935 0.802 0.065 0.667 0.761 6 0.935 0.895 0.074 0.560 0.774 7 0.924 0.833 0.152 0.585 0.753 8 0.935 0.850 0.096 0.553 0.769 9 0.945 0.885 0.140 0.600 0.820 10 0.949 0.833 0.090 0.554 0.807 11 0.936 0.802 0.048 0.651 0.770 12 0.906 0.780 0.100 0.550 0.798 13 0.942 0.800 0.155 0.590 0.700 14 0.916 0.789 0.141 0.576 0.745 15 0.952 0.839 0.144 0.506 0.755 16 0.920 0.831 0.100 0.517 0.768 17 0.924 0.818 0.128 0.550 0.760 18 0.913 0.855 0.267 0.500 0.750

fied ganglion protein corresponded to one putative locus 115) also vary considerably, but the variation seems to Ugp with two co-dominant alleles, Ugp(lOO) and have a geographic basis. Thus, the Mdh(115) allele fre­ Ugp(85). One-banded homozygotes and three-banded quency changes from 0.048 to 0.152 in the Kurile- heterozygotes suggest that the protein is a dimer. No Komandor region and equals 0.136 in the Sea of Japan, violations of the Hardy-Weinberg proportions were while in the Gulf of Alaska it equals 0.267. The Sea of registered for the protein phenotypes in any of the Japan sample differs from the other samples by the samples. comparatively low frequency of the Mdh(lOO) allele All four polymorphic loci exhibited geographic vari­ (0.460). Mean frequency of the Mdh(lOO) allele ranges ation of their common alleles. Differences in allelic from 0.547 to 0.667 among the Kurile samples, from frequencies were observed between three main geo­ 0.506 to 0.590 among the Komandor samples, and 0.500 graphic regions of the North Pacific: the Sea of Japan, in the sample from the Gulf of Alaska. Heterogeneity the Kurile-Komandor region, and the Gulf of Alaska. analysis revealed significant differences at the Mdh locus Mean frequency of the most common allele Pgd(lOO) in the Kurile samples, in the Komandor-Kurile samples, was rather invariant, ranging through the Gulf of Alaska and in all the samples taken together (Table 3). to the Komandor and Kurile Islands from 0.906 to 0.952, The Ugp locus showed some geographical pattern of and higher in the Sea of Japan, namely 0.980. Con­ variation. The frequency of the Ugp(100) allele equals tingency chi-square analysis at the Pgd locus for geo­ 0.864 in the sample from the Sea of Japan, while in the graphical groups of samples and for the whole set of other samples it ranges from 0.745 to 0.807. The overall samples did not show significant values of heterogeneity heterogeneity at the Ugp locus is apparently attributable (Table 3). to the significant distinction of the Sea of Japan squid Two common alleles at the Pgm-1 locus (100 and 105) from the others (Table 3). presented considerable variation between samples. Comparing common allele frequencies at the four Thus, sample frequency of the Pgm-1 (105) changes from polymorphic loci of females with those of males in the 0.077 to 0.172 in the central Kurile only, while in the Sea most numerous sample 4 from the central Kurile did not of Japan and in the Gulf of Alaska it equals 0.173 and reveal significant differences (Table 4). No value even 0.134 respectively, showing no geographical depen­ approached the 5% rejection level (t = 1.98; d.f. > 30). dence. Contingency chi-square analysis revealed highly F-statistics combined across all four polymorphic loci significant heterogeneity at the Pgm-1 for all the for the samples taken a over vast territory of the North samples, which is apparently attributable to the allelic Pacific oceanic slope appeared to be very small (0.004). differences between the samples of squid from the Kur­ This means that practically all genetic differentiation is ile region (Table 3). contained within samples. Hierarchical analysis using Three common alleles at the Mdh locus (7 5 ,100 and Wright’s F-statistics suggested that half of the total vari- ICES mar. sd. Symp.. 199(1995) Genetic differentiation in Berryteuthis magister from the North Pacific 463

Table 3. Contingency chi-square analysis for samples of Berryteuthis magister from different geographic areas.______

Loci Groups of Totals samples Pgd Pgm-1 Mdh UgP 1.3 49.9 Central Kuriles 4.4 27.4 16.8 X 30 (samples 3-6) d.f. 9 9 9 3 n.s. < 0.01 n.s. n.s. <0.05 3.6 44.8 Northern Kuriles 8.0 13.7 19.6 X“ 40 (samples 7-11) d.f. 12 12 12 4 n.s. n.s. n.s. n.s. n.s. 116.6 All Kuriles X 14.6 49.9 45.9 6.2 90 (samples 2-11) d.f. 27 27 27 9 n.s. < 0.01 <0.05 n.s. <0.05 13.2 4.2 40.3 Komandors X 9.5 13.4 4 40 (samples 12-16) d.f. 12 12 12 n.s. n.s. n.s. n.s. n.s. 14.2 201.3 Kuriles, and X 29.7 84.4 73.0 150 western Bering Sea d.f. 45 45 45 15 < 0.01 (samples 2-17) n.s. <0.001 < 0.01 n.s. 287.3 All samples grouped 56.1 95.1 105.3 30.8 X 170 (samples 1-18) d.f. 51 51 51 17 n.s. <0.001 < 0.001 <0.05 <0.001

d.f. = degrees of freedom; p = probability, n.s. — not significant; p > 0.05.

Table 4. Allelic differences between females and males of the squid Berryteuthis magister.

Allele

Pgd(lOO) Pgm-1 (100) Mdh(lOO) Ugp(lOO)

Frequency in females (n = 112) 0.929 0.866 0.545 0.777 Frequency in males (n = 111) 0.932 0.873 0.532 0.811

t 0.13 0.22 0.30 0.89

n = number of specimens; t = statistical significance of allelic differences between sexes.

ance (0.002) is due to differences between samples group of samples (5, 7, 11, 13, 14), but it is arguable within each geographical group, and half (0.002) to whether this pattern reflects a geographic basis of gen­ differences between geographical groups of samples. etic variation. When individual loci are taken into account, the most The pattern of genetic variation at the four loci stud­ variable locus is the Mdh with an F-value of 0.007; ied is observed when successive yearly samplings of B. Mdh(115) making the largest contribution to the total magister from the same localities are taken into account. value. Ripening animals caught near the central Kurile Islands The phenogram constructed from UPGM A clustering (samples 5 and 6) in May during two years appeared in of Nei’s genetic distances (1972) is similar to the one different clusters on both dendrograms. Such tempor­ produced from Rogers’ genetic distances (1972) (Fig. 2). ally dependent differentiation, supported by the result­ These two phenograms differ in minor rearrangements ing clustering using two different genetic distances, was of branches, but generally show the same topology. also characteristic for successively sampled squid from Thus, both trees show differentiation of Japanese (1) the north Kurile region (samples 7 and 8, samples 10 and and Alaskan (18) samples from the western Bering Sea- 11) and from the Komandor region (samples 12, 14, Kurile group of samples, which reflects the geographic and 16). Genetic differentiation as witnessed by the basis for genetic relationships of squid from remote UPGM A analysis of genetic distances is less pronounced localities. Some clustering is observed for the Kurile when sampling is made within a locality monthly samples (2, 3, 4, 6, 8, 10), for the western Bering Sea (samples 13 and 14), or during several months (samples samples (15, 16, 17), and for the Kurile-Komandor 3 and 4, samples 15 and 16). Genetic differentiation as 464 O. N. Katugin ICES mar. Sei. Symp., 199 (1995)

1 2 that the revealed electrophoretic variation is genetically 10 determined. For B. magister the enzyme patterns were consistent 4 with those mentioned by Darnall and Klotz (1975) for 15 16 another zoological taxa, suggesting that the observed 17 protein variation in this squid had a genetic basis. 12 5 Hardy-Weinberg chi-square goodness-of-fit tests for 11 three polymorphic enzyme loci and for one polymorphic non-enzyme locus in each of 18 collections yielded only ------13 one significant outcome, supporting the idea of a genetic ------9 18 basis for the variation. The only significant value, regis­ tered for the Mdh locus in the sample from the Navarin ~~l 1 I 1 I------1------1 0.06 0.05 0.04 0.03 0.02 0.01 0.00 Bay, could be due to chance, since one significant out­ ROGERS’ GENETIC DISTANCE come out of 20 tests is expected under the accepted 5% probability of rejection of the hypothesis. Wright’s Fst statistic (Wright, 1965) is an accepted estimate of genetic differentiation among populations. This measure of differentiation between sampled populations ranges from 0 to 1, and is generally higher in organisms with restricted vagility and low potential 16 for gene flow. For animals with a high extent of dispersal, such as marine fish, the F-value usually falls between 0.0041 (Pleuronectesplatessa, 5 loci; Ward and Beardmore, 1977) and 0.041 (Chanos chanos, 9 loci; Winans, 1980), though it can vary considerably due to such factors as breeding systems (Allard et al., 1968; Brown, 1979; Saura, 1983), migratory abilities (Eanes 1 1 and Koehn, 1978) and evolutionary history (Grant, 0.010 0.000 1981). NEI'S GENETIC DISTANCE Although only four polymorphic loci were included in Figure 2. Dendrograms constructed by the UPDMA from the analysis of genetic differentiation in B. magister, the Nei s (1972) and Rogers’ (1972) genetic distances for data from four polymorphic loci of Berryteuthis magister. wide geographical range of sampling represents differ­ ent habitat and biological peculiarities of this squid. We may, therefore, consider the magnitude of FST of 0.004 witnessed by the UPGMA analysis of genetic distances characteristic for this species. Such a value is apparently is less pronounced. very low, suggesting little differentiation within the species as compared to other animal taxa of outbreeders (Saura, 1983). Within-region, and between-region Discussion values of genetic differentiation are equal at 0.002, re­ flecting the fact that differences on a small scale between Biochemical genetic techniques such as electrophoresis alternating generations of squid account for the total with subsequent specific and non-specific biochemical value as much as differences on a large scale between staining have provided a useful tool for assessing intras­ geographically isolated populations. pecific differentiation in a number of commercial Analysis of genetic distances between samples of B. species, mostly fishes (Allendorf and Utter, 1979; Berst magister along with analysis of allelic heterogeneity and Simon, 1981; Shaklee, 1983). The genetic basis of among the samples supported the hypothesis of small the observed variation in proteins has been established but significant intraspecific genetic differentiation. Pair­ either through inheritance tests (see Allendorf and wise D n values were rather low, of 0.001-0.016, but Utter, 1979; Kornfield et al., 1981), or based on indirect they were obtained from four polymorphic loci only, and criteria such as agreement of banding patterns of pre­ not really comparable with those obtained for other sumed heterozygotes for a particular protein with those animal taxa on large samples of genetic loci (Ayala, from another taxa (Darnall and Klotz, 1975; Harris and 1975, 1982; Nei, 1987). Nevertheless, we were able to Hopkinson, 1976). Good agreement between the ob­ assess whether different estimates of genetic distances served and expected genotypic frequencies, as tested by adequately discriminate between samples of B. magis­ the chi-square statistic, usually supports the hypothesis ter, and whether that differentiation could be explained Genetic differentiation in Berryteuthis magister from the North Pacific 465

Table 5. Biological and genetic characteristics of Berryteuthis magister from different regions of the North Pacific.

Mean allele frequencies Time of Ugp(lOO) Region DMLab r , c a spawningab Pgd(lOO) Pgm-1 (100) Mdh(115) 0.864 Sea of Japan 20-22 0.3-0.5 winter 0.980 0.797 0.136 0.782 Kurile Chain 26-28 30-3.5 summer 0.935 0.847 0.092 0.754 Western Bering Sea 26-28 3.0-3.5 summer 0.927 0.810 0.128 0.750 Gulf of Alaska 28-32 4.0-5.0 spring 0.913 0.855 0.267

DML = average dorsal mantle length of mature females (cm). T° = average temperature at which squid is most likely to live, a = data, kindly presented by Dr G. A. Shevtsov and Dr Yu. A. Fedorets. b = Yuuki and Kitazawa (1986).

on geographical and life history scales. Similar trees and Kitazawa, 1986). The largest animals are found in obtained from both D N and D R values support the idea the Gulf of Alaska (Shevtsov and Fedorets, pers. of geographic pattern in the variation in squid: both comm.). Squid generally spawn in winter with a peak Japanese and Alaskan samples are clearly distinguished between February and March in the Sea of Japan (Yuuki from the others. Phenetic topology obtained for 16 and Kitazawa, 1986), while in other regions the spawn­ samples from the Northwest Pacific did not reveal a ing period is generally restricted to spring and summer geographic pattern of variation, but could be due to (Naito et al., 1977; Fedorets, pers. comm.). Prespawn­ some differences between successive generations of ing females from the Sea of Japan are characterized by squid from the Kurile area. larger eggs than those from the Bering Sea, and the Gulf B. magister is considered to have several spawning of Alaska (4.2-5.9 mm versus 3.5 mm in diameter) areas. Though spawning or newly laid eggs have never (Nesis, 1989; personal observations). been seen by scientists, the locations of the spawning Large-scale differentiation of the B. magister gene grounds have been determined from the presence of pool into three discrete parts is therefore concordant hatchlings (paralarvae), and epiplanktonic juveniles. with data on the life cycle of the species, and with the After hatch somewhere on the continental slope, pre­ geographical distribution of its life stages. Geographical sumably in early spring, young paralarvae lead plank- and hydrological barriers between the Sea of Japan, the tonic lives in the epipelagic layers until the beginning of Northwest Pacific, and the Gulf of Alaska, therefore onthogenetic descent in summer or early fall (Kubo- could account for small though significant spatial allelic dera, 1982). Pelagic paralarvae and juveniles may be differentiation in the squid. Intersample genetic differ­ free-swimming for approximately five months. Squid in entiation is usually weakly pronounced in marine neritic early life stages are transported by the North Pacific species with wide distribution ranges, large population currents along the continental shelf and chains of sizes, and high vagility (e.g., Andersson et al., 1981; islands, such as the Kurile and Aleutian Archipelagos, Grant, 1987; Winans, 1980; Winans, unpublished data). concentrating in several regions: in the Sea of Japan, Two peaks of spawning registered for B. magister in near the southwestern Kamtchatka in the Sea of the western Bering Sea, particularly, near the Koman­ Okhotsk, in the south central Bering Sea, and in the dor Islands (Fedorets, pers. comm.) could account for Gulf of Alaska (Kubodera and Jefferts, 1984; Yuuki and temporal differences in allelic frequencies observed for Kitazawa, 1986; Okutani, 1988). Long-term passive mi­ the squid from the Komandor-Kurile area. Unexpec­ grations of newly hatched squid together with spatial tedly small genetic differences were found between two isolation of spawners may have provided biological and temporally and spatially isolated stocks of Loligo gahi, geographical barriers for gene flow between populations spawning in spring and in summer, which led the authors of B. magister from the Sea of Japan, the Kurile-western to assume one panmictic population of this squid in Bering Sea area, and the Gulf of Alaska, which resulted Falkland waters (Carvalho and Pitcher, 1989). Tem­ in the observed patterns of allele frequency distribution poral allele frequency shifts within a geographic area at polymorphic loci, and also in certain biological differ­ have been found for several marine fishes (Kornfield et ences (Table 5). a i, 1981; Gyllensten and Ryman, 1988), but no reason­ B. magister from different regions have been recog­ able explanation provided. Minor differences in genic nized by a number of biological characters. Fully mature characters observed in the Kurile-western Bering Sea specimens from the Sea of Japan are, on average, region could be due to some selectional forces that smaller than those from other parts of the range (Yuuki favour genotypes adapted for certain micro-scale con- 466 O. N. Katugin ICES mar. Sei. Sy mp., 199 (1995)

di tions for growth of early life stages. A similar expla­ Allendorf, F. W., and Utter, F. M. 1979. Population genetics. nation was suggested for the pattern of genetic differen­ In Fish physiology. Vol. 8. pp. 407-454. Ed. by W. S. Hoar, tiation of the Atlantic eel, where the species probably D. J. Randall, and J. R. Brett. Academic Press, New York. Ally, J. R. R., and Keck, S. C. 1978. A biochemical genetic consists of one large panmictic gene pool and the ob­ population structure study of the market squid, Loligo opa- served changes in allelic frequencies are due to natural lescens, along the California coast. Calif. Dept. Fish. Game selection rather than to population structuring (Koehn Fish. Bull., 169: 113-121. and Williams, 1978). It is worth mentioning that oc­ Andersson, L., Ryman, N., Rosenberg, R., and Stahl, G. 1981. Genetic variability in Atlantic herring (Clupea harengus har- casionally prespawning B. magister have been caught engus): description of protein loci and population data. Her- over the entire Kurile area, suggesting that the spawning editas, 95: 69-78. grounds are not completely restricted to one particular Augustyn, C. J., and Grant, W. S. 1988. Biochemical and locality and that there is potential for gene flow in the morphological systematics of Loligo vulgaris vulgaris region consistent with the idea of natural selection caus­ Lamarck and Loligo vulgaris reynaudii d’Orbigny nov. comb. (Cephalopoda: Myopsida). Malacologia, 29: 215- ing the observed genetic differentiation between tem­ 233. poral groups of B. magister. On the other hand, minor Ayala, F. J. 1975. Genetic differentiation during the spéciation genetic differentiation of the squid within the geo­ process. Evol. Biol., 8: 1-78. graphic region could be due to the existence of several Ayala, F. J. 1982. Population and evolutionary genetics. A primer. Benjamin/Cummings. Menlo Park, California. temporally and (or) spatially isolated stocks, or sub- Berst, A. H., and Simon, R. C. (eds.) 1981. Proceedings of the populations, and further investigation is needed to solve Stock Concept International Symposium. Can. J. Fish, the problem, perhaps using more sensitive microsatellite aquat. Sei., 38: 1309-1921. or mtDNA sequence methods. Brierley, A. S., Rodhouse, P. G., Thorpe, J. P., and Clarke, M. R. 1993a. Genetic evidence of population heterogeneity and cryptic spéciation in the ommastrephid squid Martialia Conclusion hyadesi from the Patagonian Shelf and Antarctic Polar Fron­ tal Zone. Mar. Biol., 116(4): 593-602. Two main factors apparently contribute to the observed Brierley, A. S., Thorpe, J. P., Clarke, M. R., and Martins, H. R. 1993b. A preliminary biochemical genetic investi­ patterns of genetic differentiation in the gonatid squid gation of population structure of Loligo forbesi Steenstrup, B. magister. These are: (1) population genetic diver­ 1856 from the British Isles and the Azores. In Recent ad­ gence during separate evolution of squid from three vances in cephalopod fisheries biology, pp. 61-69. Ed. by T. geographically distinct regions of the Subarctic Pacific: Okutani, R. K. O’Dor, and T. Kubodera. Tokai University Press, Tokyo. the Sea of Japan, the Northwest Pacific (the Kurile Brown, A. H. D. 1979. Enzyme polymorphism in plant popu­ Chain, western part of the Aleutian Archipelago, the lations. Theor. Pop. Biol., 15: 1-42. western Bering Sea), and the Gulf of Alaska; and (2) Carvalho, G. R., and Loney, K. H. 1989. Biochemical genetic minor genetic differences acquired between successive studies on the Patagonian squid Loligo gahi d’Orbigny. I. generations of squid inside at least one macrogeographic Electrophoretic survey of genetic variability. J. Exp. Mar. Biol. Ecol., 126(3): 231-241. locality, the Northwest Pacific, attributed either to Carvalho, G. R., and Pitcher, T. J. 1989. Biochemical genetic natural selection on early life stages or to isolation be­ studies on the Patagonian squid Loligo gahi d’Orbigny. II. tween successive breeding units. Population structure in Falkland waters using isozymes, mor­ phometries and life history data. J. Exp. Mar. Biol. Ecol 126(3): 243-258. Acknowledgements Carvalho, G. R., Thompson, A., and Stoner, A. L. 1992. Genetic diversity and population differentiation of the short- I sincerely appreciate the generous help of Dr Yuri fin squid Illex argentinus in the Southwest Atlantic. J. Exp. Fedorets (TINRO, Vladivostok), who provided frozen Mar. Biol. Ecol., 158: 105-121. Christofferson, J. P., Foss, A., Lambert, W. E., and Welge, B. samples of B. magister for electrophoretic investigation, 1978. An electrophoretic study of select proteins from the and also allowed me to use his unpublished data on squid market squid, Loligo opalescens, Berry. Calif. Dept. Fish biology. I am grateful to Dr Vladimir Efremov (Institute Game Fish. Bull., 169: 123-133. of Marine Biology, Vladivostok) for help in analysing Darnell, D. W., and Klotz, I. M. 1975. Subunit composition of gene frequency data. Useful consultations with Dr Gen­ proteins: a table. Arch. Bioch. Bioph., 166: 651-682. Eanes, W. F., and Koehn, R. K. 1978. An analysis of genetic nadi Shevtsov (TINRO, Vladivostok) and Dr Alex­ structure in the monarch butterfly, Danaus plexippus L ander Pudovkin (Institute of Marine Biology, Vladivos­ Evolution, 32: 784-797. tok) are acknowledged. Fedorets, Yu. A. (in press) On size-sexual structure of the squid Berryteuthis magister from the Komandor and the Kurile Islands. (In Russian.) References Garthwaite, R. L., Berg, Jr. C. J., and Harrigan, J. 1989. Population genetics of the common squid Loligo pealei Allard, R. W., Jain, S. K., and Workman, P. L. 1968. The LeSueur, 1821, from Cape Cod to Cape Hatteras. Biol. Bull genetics of inbreeding populations. Adv. Genet., 14: 55- (Woods Hole), 177: 287-294. 131. Grant, W. S. 1981. Biochemical genetic variation, population ic e s mar sd. Symp.. 199 (1995) Genetic differentiation in Berryteuthis magister from the North Pacific 467

structures, and evolution of Atlantic and Pacific herring. Legkaya i Pischevaya Promyshlennost Publ. Moscow. 358 PhD Thesis, University of Washington, Seattle. 135 pp. pp. (In Russian.) Grant, W. S. 1987. Genetic divergence between congeneric Nesis, K. N. 1989. Teuthofaunaof the Okhotsk Sea. Biology of Atlantic and Pacific Ocean fishes. In Population genetics and gonatid squids Berryteuthis magister and Gonatopsis borea­ fisheries management, pp. 225-246. Ed. by N. Ryman and F. lis. Zool. J., 68(9): 45-56. (In Russian.) Utter. University of Washington, Seattle. Okutani, T. 1988. Evidence of spawning of Berryteuthis magis­ Gyllesten, U., and Ryman, N. 1988. Biochemical genetic vari­ ter in the northeastern Pacific. Bull. Ocean Res. Inst. Univ. ation and population structure of fourhom sculpin (Myoxo- Tokyo, 26(1): 193-200. cephalus quadricornis; Cottidae) in Scandinavia. Hereditas, Peacock, A. C., Bunting, S. L., and Queen, K. G. 1965. Serum 108: 179-185. Protein electrophoresis in acrylamide gel: patterns from Harris, H., and Hopkinson, D. A. 1976. Handbook of enzyme normal human subjects. Science, 147: 1451-1455. electrophoresis in human genetics. Elsevier, North-Holland, Rogers, J. S. 1972. Measures of genetic similarity and genetic Amsterdam, 350 pp. distance. Studies in Genetics, Univ. Texas Publ., 72-13: Katugin, O. N. 1991. Unidentified gangle protein polymor­ 145-153. phism in the gonatid squid Berryteuthis magister from the Saura, A. 1983. Population structure, breeding systems and North Pacific. Isozyme Bull., 24: 51. molecular taxonomy. In Protein Polymorphism: Adaptive Kim, Y. H. 1993. Population analysis of the common squid, and Taxonomic Significance, pp. 301-324. Ed. by G. S. Todarodes pacificus Steenstrup in Korean waters. PhD The­ Oxford and D. Rollinson, Academic Press, London. sis. Dept. Mar. Biol. Graduate School Natnl. Fish. Univ. Shaklee, J. B. 1983. The utilization of isozymes as gene markers Pusan. 106 pp. (In Korean with English summary.) in fisheries management and conservation. In Isozymes. Cur­ Koehn, R. K., and Williams, G. C. 1978. Genetic differen­ rent Topics in Biological and Medical Research. Vol. 11. pp. tiation without isolation in the American eel, Anguilla 213-247. Ed. by M. Rattazzi, J. G. Scandalios, and G. S. rostrata. II. Temporal stability of geographical patterns. Whitt. Alan R. Liss, Inc., New York. Evolution, 32: 624-637. Shaw, C. R., and Prasad. R. 1970. Starch gel electrophoresis of Kornfield, I., Gagnon, P. S., and Sidell, B. J. 1981. Inheritance enzymes - a compilation of recipes. Biochem. Genet., 4: of allozymes in Atlantic herring (Clupea harengus harengus). 297-320. Can. J. Genet. Cytol., 23: 715-720. Smith, P. J., Mattlin, R. H., Roeleveld, M. A., and Okutani, Kubodera, T. 1982. Ecological studies of pelagic squids in the T. 1987. Arrow squids of the genus Nototodarus in New Subarctic Pacific region. Doctoral Diss., Hokkaido Univ. Zealand waters: systematics, biology, and fisheries. N.Z. J. Hakodate. 225 pp. (In Japanese). Mar. Freshwat. Res., 21: 315-326. Kubodera, T., and Jefferts, K. 1984. Distribution and abun­ Sokal, R. R., and Michener, C. D. 1958. A statistical method dance of the early life stages of squid, primarily Gonatidae for evaluating systematic relationships. Univ. Kansas Sei. (Cephalopoda, Oegopsida) in the northern North Pacific. Bull., 28: 1409-1438. Bull. Natn. Sei. Mus. Tokyo, ser A, 10: 91-106,165-193. Urbach, V. Yu. 1964. Biometric methods. Nauka Publ. Mos­ Murata, M., Ishii, M., and Kubota, S. 1985. Some consider­ cow. 415 pp. (In Russian.) ations on population structure of flying squid, Ommastrephes Ward, R. D., and Beardmore, J. A. 1977. Protein variation in bartrami (LeSueur), in the North Pacific. Report of 1983 the plaice Pleuronectesplatessa L. Genet. Res., 30: 45-62. Annual Meeting on Resources and Fisheries on Squids. Winans, G. A. 1980. Geographic variation in the milkfish Tohoku Reg. Fish. Res. Lab. (Hachinohe Branch): 6-49 (in Chanos chanos. I. Biochemical evidence. Evolution, 34: Japanese). 558-574. Naito, M., Murakami, K., and Kobayashi, T. 1977. Growth Workman, P. L., and Niswander, J. D. 1970. Population and food habit of oceanic squids (Ommastrephes bartrami, studies on southwestern Indian tribes. II. Local genetic dif­ Onychoteuthis borealijaponica, Berryteuthis magister, and ferentiation in the Papago. Am. J. Human Genet., 22: 24- Gonatopsis borealis) in the western Subarctic Pacific region. 49. Res. Inst. N. Pac. Fish., Fac. Fish., Hokkaido Univ. Sp., p. Wright, S. 1965. The interpretation of population structure by 339-351. (In Japanese with English abstract.) F-statistics with special regard to systems of mating. Evol­ Natsukari, Y., Nishiyama, Y., Nakanishi, Y. 1986. A prelimi­ ution, 19: 395-420. nary study on the isozymes of the loliginid squid, Photololigo Wright, S. 1978. Evolution and the Genetics of Populations, edulis (Hoyle, 1885). Rep. Coop. Invest. Shiro-Ika, Loligo Vol. 4. Variability within and Among Natural Populations. edulis, inhabited western Japan Sea, 2: 145-151. Univ. Chicago Press, Chicago. Nei, M. 1972. Genetic distance between populations. Am. Yeatman, J. M ., and Benzie, J. A. H. 1993. Cryptic spéciation Nat., 106: 283-292. in Loligo from Northern Australia. In Recent advances in Nei, M. 1977. F-statistics and analysis of gene diversity in cephalopod fisheries biology, pp. 641-652. Ed. by T. Oku­ subdivided populations. Ann. Human. Genet., 41: 225-233. tani, R. K. O’Dor, andT. Kubodera. Tokai University Press, Nei, M. 1987. Molecular evolutionary genetics. Columbia Tokyo. Univ. Press. New York. 496 pp. Yuuki, Y., and Kitazawa, H. 1986. Berryteuthis magister in the Nesis, K. N. 1973. Systematics, phylogeny, and evolution of the southwestern Japan Sea. Bull. Japan. Soc. Sei. Fish., 52(4): gonatid squids. Zool. J., 52(11): 1626-1638. (In Russian.) 665-672. Nesis, K. N. 1982. Short guide to cephalopods of the world.